Biopolymer System for Tissue Sealing

ABSTRACT

A tissue sealant for use in surgical and medical procedures for sealing the tissues of a living mammal is provided. The tissue sealant comprises a hydrogel which is formed by gelation of a premix disposed on the tissue to be sealed. The premix comprises alkylated chitosan or a gelatin, and a polybasic carboxylic acid or an oxidized polysaccharide, in an aqueous medium. The premix can also include a dehydrating reagent, a carboxyl activating reagent, or both. A specific use of the tissue sealant is in the repair of the dura mater after brain surgery to prevent leakage of cerebrospinal fluid. The tissue sealant may include a therapeutic or protective agent such as an antibiotic or an anti-inflammatory drug.

CLAIM OF PRIORITY FROM A PRIOR-FILED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/379,182, filed Apr. 18, 2006, which is incorporated hereinby reference, and claims the priority thereof.

FIELD OF THE INVENTION

The invention relates to tissue sealants for medical or veterinary use,methods of preparing the sealants, and methods of using them in medicalprocedures.

BACKGROUND OF THE INVENTION

Tissue sealants are increasingly important adjuncts in surgicalprocedures, being used in fields such as vascular surgery, cardiacsurgery, spine surgery and brain surgery as well as in general surgery.Uses for tissue sealants include, among others, augmenting or replacingsutures to join tissues or place them in proximity, closing perforationsin biological membranes to prevent leakage of fluids, incorporatingmedicinal substances at the location of emplacement for localizedrelease, and filling areas of tissue removal. One commonly used tissuesealant is fibrin glue, a material analogous to clotted blood, which isobtained from reaction of fibrinogen and thrombin isolated from bloodplasma. For example, see “Fibrin Glue from Stored Human Plasma: AnInexpensive and Efficient Method for Local Blood Bank Preparation,”William D. Spotnitz, M.D., Paul D. Mintz, M.D., Nancy Avery, M.T.,Thomas C. Bithell, M.D., Sanjiv Kaul, M.D., Stanton P. Nolan, M.D.(1987), The American Surgeon, 53, 460-62. However, concern aboutpossible viral or prion contamination of human blood-derived proteinproducts, and dissatisfaction with the relatively long times oftenrequired for fibrin gelation or “setting” to occur, have resulted in asearch for tissue sealants with more advantageous properties.

There have been systems developed that use fibrin glues as part of amore complex assembly with more favorable properties. U.S. Pat. No.6,699,484 discusses the use of fibrinogen in mixtures withpolysaccharides such as hyaluronan and chitosan to form surgicaladhesives, wherein the fibrinogen and thrombin components aredistributed in dry form on a support comprising the polysaccharide,which is activated by water when emplaced on a wound to form a sealant.

In an attempt to avoid the use of human blood products, other mammaliansources of proteins have been studied. A tissue sealant has beenprepared using bovine serum albumin that is crosslinked withglutaraldehyde. An example is BioGlue Surgical Adhesive® produced byCryoLife, Inc. of Kennesaw, Ga. However, bovine tissues are also asource of concern in terms of the possible presence of pathogenicentities such as viruses or prions. The types of processing required todestroy viruses or prions also tend to denature the desired proteins andmake them intractable as sealants.

A tissue sealant that does not use proteins isolated from mammalianblood, such as Duraseal® produced by Confluent Surgical Inc. of Waltham,Mass., comprises tri-lysine-amine and an activated polyethyleneglycol. Asimilar product, termed CoSeal® and produced by Baxter of Deerfield,Ill., is likewise composed of synthetic functionalizedpolyethyleneglycol derivatives, also avoiding the use of blood-derivedmaterials. However, both of these synthetic hydrogels are dimensionallyunstable in the presence of water, undergoing considerable swelling. Forexample, see “Evaluation of Absorbable Surgical Sealants: In vitroTesting,” Patrick K. Campbell, PhD, Steven L. Bennett, PhD, ArtDriscoll, and Amar S. Sawhney, PhD, atwww.duralsealant.com/duralsealant/literature.htm (as of Aug. 24, 2006).This tendency to swell can be highly disadvantageous in certainapplications, such as neurosurgery, where the resulting pressure onnerve or brain tissue can produce serious side-effects.

Chitin, a biopolymer that is abundant in the shells of arthropods, is aβ-1,4 polymer of 2-acetamido-2-deoxyglucose. During its isolation, it isfreed from proteinaceous and mineral components of the shell. Purifiedchitin can be further processed by chemical treatment resulting indeacetylation to yield chitosan, (poly-(2-amino-2-deoxyglucose)), whichis a basic (alkaline) substance due to its free amino groups. From theperspective of medical uses, chitosan offers several desirableproperties. The material is known to be non-toxic and biocompatible, andsince chitin is not derived from vertebrates and is processed underrather harsh conditions such as exposure to alkalai during itstransformation into chitosan, the possibility of contamination withviruses or prions that are pathogenic to mammals is very low. Theutility of biocompatible chitosan derivatives in medical applicationshas received attention. For example, U.S. Pat. No. 5,093,319 discussesthe use of films prepared from carboxymethylated chitosan for use insurgery to prevent post-operative adhesion of injured soft tissues uponhealing. The chitosan derivatives are described to be formed into abiodegradable “sheet” that during surgery is emplaced between softtissues for which adherence during healing is not desired. In anothertype of use, U.S. Pat. No. 4,532,134 discusses the use of chitosan inpromoting blood coagulation in wounds.

Hydrogels are gels in which water is the dispersion medium. A commonexample of a hydrogel is a gel formed from the protein gelatin in water.Other hydrogels are formed by polysaccharides such as agar dispersed inwater. Hydrogels in the form of sheets are used as wound dressings,where they are favored for their ability to help maintain a moistenvironment to facilitate healing of the wound without drying andcracking of tissues. For example, seewww.medicaledu.com/hydrogellsheet.htm. Chemical derivatives of chitosanhave also been used to form hydrogels for use as surgical sealants andin drug delivery devices. U.S. Pat. No. 6,602,952, assigned toShearwater Corp., describes the preparation ofpoly(alkyleneoxide)chitosan derivatives and their use in the formationof hydrogels. The addition of these hydrophilic but non-ionic groups tothe chitosan molecule alters its physical properties.Poly(alkyleneoxides) such as poly(ethyleneoxide), also known (somewhatinaccurately) as poly-ethyleneglycols or PEGs, are formed by thepolymerization of alkylene oxides (epoxides) such as ethylene oxide.They may be obtained in a wide variety of molecular weights, withvarious structural features such as activated end groups, hydrolysablelinkages, and others. For example, see the Nektar PEG catalog that listsa wide variety of the Shearwater functionalized PEGs, atwww.nektar.com/pdf/nektar_catalog.pdf (as of Aug. 24, 2006).

Other methods have been described for the preparation of hydrogels fromchitosan. The published PCT application WO2005/113608 and the publishedU.S. patent application no. 2005/0271729, both by the same inventor,discuss the crosslinking of chitosan and hyaluronan, also known ashyaluronic acid. Hyaluronan is an acidic linear polysaccharide formed ofβ-1,3 linked dimeric units, the dimeric units consisting of an2-acetamido-2-deoxyglucose and D-gluconic acid linked in a β-1,4configuration. These published applications discuss crosslinking the twotypes of polysaccharides using a carbodiimide reagent.

Hydrogels comprising chitosan derivatives and polybasic carboxylic acidsor oxidized polysaccharides, for use in vascular occlusion, are alsodisclosed in copending U.S. patent application Ser. No. 11/425,280,filed Jun. 20, 2006 by the same inventors as in the present application.

There is an ongoing need for a hydrogel tissue sealant that is not bloodor animal protein derived, that consists of biocompatible materials, isdimensionally stable after emplacement in the patent's body, has goodsealant and tissue adhesive properties, is of sufficient strength andelasticity to effectively seal biological tissues, that can be readilyprepared and used during surgery, and that forms the tissue seal on atimescale compatible with surgery on living patients.

SUMMARY OF THE INVENTION

The present invention provides a tissue sealant for medical orveterinary use in repair of physical damage to living mammalian tissuessuch as cuts, tears, holes, bone breaks and other unintentional injury.

The invention further provides a tissue sealant for medical orveterinary use in repair of physical damage resulting from surgicalprocedures, such as in closing a suture line, reinforcing a suture line,tissue approximation using the sealant instead of a suture, filling adisused dead space or void, or sealing a vascular defect.

The invention further provides a tissue sealant useful in medicalprocedures such as in preventing post-surgical adhesions, as a mechanismof drug delivery, or in coating implanted medical devices.

The invention further provides a tissue sealant that is well-suited forthe repair and sealing of membranous biological tissues, in particularthe dura mater and other membranes surrounding neural tissue.

The invention further provides a tissue sealant that due to itsexceptional dimensional stability may be used in situations whereswelling and the resulting pressure are undesirable and produce unwantedside effects.

The invention further provides a tissue sealant that offers a very lowrisk of contamination by pathogens such as viruses and prions.

The invention further provides a tissue sealant that is not preparedfrom human blood products, which is desirable because human bloodproducts carry a risk of contamination with pathogens and are alsoobjectionable to certain patients on religious and moral grounds.

The invention further provides a premix, a flowable, substantiallyliquid sol, that on standing forms a hydrogel that seals biologicaltissues, preferably adhering to the tissues.

A composition of the invention can comprise a chitosan derivative thathas been modified by the introduction of covalently bound moieties ontothe polymer chain. The chitosan derivative, and a polybasic carboxylicacid or an oxidized polysaccharide, upon dissolution in an aqueousmedium can initially form a flowable, substantially liquid sol, apremix, that over a period of time, typically in the order of minutes,gels to form a hydrogel of the invention. The hydrogel, which isbiocompatible and can be biodegradable, when formed serves to sealtissues that have been cut or torn, for example from a surgicalprocedure or from an injury. The hydrogel can also adhesively sealtissues.

The invention further provides an embodiment of a premix comprising apoly(oxyalkylene)chitosan and a hyaluronan. In another embodimentaccording to the present invention, the premix comprises an acrylatedchitosan and a dibasic carboxylic acid.

The invention further provides a premix for a hydrogel tissue sealantcomprising an alkylated chitosan, a polybasic carboxylic acid, acarboxylic acid activating reagent, a dehydrating reagent, and anaqueous medium. The carboxyl activating reagent can be an N-hydroxycompound that can form an ester with the carboxyl group, preferablyN-hydroxysuccinimide (NHS) or N-hydroxybenztriazole. The dehydratingreagent can be a carbodiimide that removes the elements of water fromreactants by thermodynamically driving the reaction, through formationof a urea compound. In one embodiment, the specific carbodiimide is1-ethyl-3-(N,N-dimethylpropyl)carbodiimide (EDCI).

The invention also provides an embodiment of a flowable aqueous solcomprising a gelatin and an oxidized polysaccharide. A specific exampleis a composition comprising gelatin and oxidized hyaluronan.

The invention further provides methods for preparing the tissue sealantsas are described herein for medical or veterinary use. The tissuesealants comprise a hydrogel that preferably adheres to the biologicaltissue of a living mammal. A specific method of preparation of a tissuesealant comprising a hydrogel according to the present invention isthrough combination of an alkylated chitosan, and a polybasic carboxylicacid or an oxidized polysaccharide in an aqueous medium. Optionally, adehydrating reagent, a carboxyl-activating reagent, or both, may beincluded.

The invention further provides methods for using a hydrogel according tothe present invention in tissue repair and other medical procedures. Aspecific embodiment of a hydrogel according to the present invention isused to reinforce a suture line, or to seal cut, torn, or perforatedtissues. It is also used to prevent leakage of biological fluids, suchas cerebrospinal fluid, through repair of biological membranes that whenintact contain the fluids. It is used to bring tissues intoapproximation and hold them in place after a surgical procedure has beencarried out.

In another specific embodiment of a use of a hydrogel according to thepresent invention, the hydrogel may further comprise a protective ortherapeutic material or substance. The substance may be an antibiotic,an anticancer agent, a peptide, a protein, a nucleic acid or a nucleicacid analog, a radioactive material, or another protective ortherapeutic substance where it is advantageous to provide the substanceat the location within the body where the hydrogel is emplaced.

For example, the protein may be a growth factor, such as a vasculargrowth factor or a factor that induces a particular kind of tissuegrowth, such as bone morphogenic factor. In another specific embodiment,the protein may be an inhibitory factor, such as a receptor antagonistsuch as for a growth factor, when supply of an inhibitory factor isdesirable, for example after removal of a tumor or cancerous tissue.

In yet another specific embodiment, the nucleic acid may be an antisensenucleic acid, or a small interfering nucleic acid analog, wherein it isadvantageous to securely emplace the material at a particular sitewithin a living mammal undergoing treatment for a condition responsiveto such therapy.

In another specific embodiment, the therapeutic agent may be anantibiotic to inhibit bacterial infection after repair of a wound orafter damage to tissues caused by surgery. Or, a protective agent may bean anti-inflammatory substance wherein it is advantageous to supply thesubstance directly at the site of damage that is repaired with thetissue sealant, such as to reduce swelling and resulting pressure onsurrounding tissues.

In another specific embodiment, the hydrogel comprises a dye, such as avisible dye or a radio-opaque dye, to enable visualization of theposition of localization of the hydrogel in the body.

In another specific embodiment, the hydrogel comprises a microsphere ora nanosphere, preferably a large number of microspheres or nanospheresdispersed in the hydrogel. Preferably the microsphere or nanospherecontains a therapeutic agent or a protective agent.

Thus, the hydrogels of the present invention, and their uses as tissuesealants, as media further containing therapeutic or protective agents,and as tissue sealants further containing therapeutic or protectiveagents, offer outstanding advantages of ease of use, biocompatibilityand biodegradability, suitability for use in conjunction with othersurgical procedures, strength, adhesivity, and versatility.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of a segment of a PEG-chitosanmolecule wherein the degree of substitution with the PEG group is 0.5.

FIG. 2 shows the chemical structure of a segment of a hyaluronanmolecule.

FIG. 3 shows the chemical structure of a segment of the PEG-chitosan ofFIG. 1 and a segment of the hyaluronan of FIG. 2 crosslinked by amidelinkages between a hyaluronan carboxylate moiety and a PEG-chitosanamino moiety.

FIG. 4 shows a segment of an acrylated chitosan polymer.

FIG. 5 shows a segment of the acrylated chitosan polymer of FIG. 4crosslinked by amide linkages formed with a adipic acid.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “tissue” refers to the material forming the solid orsemi-solid structures that make up any of the organs or components of aliving organism. Thus, liquids such as blood are not “tissue” accordingto the definition used herein, but the term “tissue” encompassesmembranes, skin, muscles, bones, cartilage, nerves and nerve sheathes,meninges, connective tissue, blood vessels, the sclera or iris of theeye, the solid materials constituting internal organs such as liver,stomach, pancreas, intestine, kidney, thymus, uterus, testes, bladder,lung, heart and any other internal structures that are solid orsemi-solid in texture.

As used herein, the term “to seal” or “sealing” refers to the actwherein two physically noncontiguous tissues or portions thereof arejoined together, or where a hole, tear, cut, perforation or otherdiscontinuity is repaired so as to close the hole, tear, cut orperforation. Sealing implies at least some degree of adhesion of thematerial used to the tissue to which it is applied, such that the sealedtissue is secured against at least a moderate displacing force. Thediscontinuity in the tissue that is being sealed may be an incision madeas part of a surgical procedure, or it may be a wound. A “sealant” is amaterial which is used to seal tissue. As mentioned, a sealant adheres,at least to some degree, to the tissue which is being sealed, such thatthe sealant material is unlikely in the short term to detach from therepaired or sealed tissue under the influence of at least a moderateforce, such as may be experienced when a patient to whom the sealant hasbeen applied moves in a normal fashion. The sealant may be biodegradableand eventually dissolve or be absorbed into the patient's body withoutdeparting from the principles of the invention.

The degree of force that is needed to disrupt a seal formed according tothe invention may vary. If the tissue is “sealed”, as the term is usedherein, the degree of adhesivity may be relatively low, such that thesealant serves to fill a void in the tissue or to keep the tissueportions in conjunction when they are not subject to a high degree ofstrain. If a tissue is “adhesively sealed,” as the term is used herein,a higher degree of strain is necessary to disrupt the bond between thetissue portions, such that rupture of the seal only takes place when arelatively high degree of force is applied. Thus, a tissue may be sealedsuch that the joined tissue portions are held in proximity by a sealantbut application of a relatively high degree of strain would tend toseparate the portions and rupture the seal, or the tissue may beadhesively sealed such that a relatively high degree of strain is neededto rupture the seal.

“Adhere” or “adherence” refers to the creation of a physical bondbetween the material and tissue such that a moderate motion or forcedoes not cause separation of the material from the tissue on which it isdisposed. Thus, a tissue sealant serves to glue together living tissue,at least temporarily, such as for the amount of time it takes healing tooccur. However, sealing may take place for a more prolonged periodwithout departing from the principles of the invention. The physicalbond that is created between the material and the tissue that is beingsealed may have one or several bases including electrostatic bonding andcovalent bonding, but any mechanism by which the adherence takes placefalls within the definition herein.

The terms “adhesive” and “adhesivity” similarly refer to the existenceof a physical bond between two materials such as a tissue sealant andthe tissue to which the sealant is applied. An adhesive is a materialwhich adheres to tissue or other material and which may be used toconstrain the separation of two tissue masses. Adhesivity is theproperty or degree to which a material adheres to a tissue or othermaterial. As used herein, adhesive tissue sealants are those sealants ofthe invention that are adapted to hold the tissue portions being sealedtogether against a relatively high degree of rupturing strain.

As used herein, a “hydrogel” refers to a material of solid or semi-solidtexture that comprises water. Hydrogels are formed by athree-dimensional network of molecular structures within which water,among other substances, may be held. The three-dimensional molecularnetwork may be held together by covalent chemical bonds, or by ionicbonds, or by any combination thereof. A common example of a hydrogel isgelatin, a protein, that “sets up” or forms a gel from a sol uponheating and subsequent cooling. Not all substances that form hydrogelsare proteins; polysaccharides such as starches may also form hydrogels.Still other hydrogels may be formed through the mixture of two or morematerials that undergo chemical reactions with each other to create thethree-dimensional molecular network that provides the hydrogel with adegree of dimensional stability. Such mixtures of materials thatinteract or react with each other to form a hydrogel are referred toherein as a “premix.” Thus, a “premix” as used herein refers to amixture of materials that after mixing will gel, or “set up,” to formthe hydrogel. A premix may be of a liquid or semi-liquid texture suchthat it can be pumped or transferred by the methods usually used forliquids, such as flow through tubes.

The act of “gelation” refers to the formation of a gel from a sol. Insome cases, the sol may consist of a single material dispersed in asolvent, typically water, as in the case of gelatin. In other cases, thesol may consist of more than a single material dispersed in a solventwherein the several materials will eventually react with each other toform a gel, and when the solvent in which they are dispersed compriseswater, the gel is a hydrogel. The hydrogels claimed herein are of thetype that are formed by the mixture of more than a single component.

A “saccharide” as used herein refers to a carbohydrate. The term“carbohydrate” includes the class of compounds commonly known as sugars,in addition to compounds that are chemically related to sugars. The termthus includes simple “monosaccharide” sugars, “disaccharide” sugars aswell as polymeric “polysaccharides.”. The term encompasses a group ofcompounds including sugars, starches, gums, cellulose andhemicelluloses. The term further encompasses sugar derivatives such asamino-sugars, for example, 2-amino-2-deoxyglucose, as well as theiroligomers and polymers; sulfated sugars; and sugars with hydroxyl,amino, carboxyl and other groups.

A carbohydrate as defined herein comprises sugars or sugar derivativeswith beta (β) or alpha (α) anomeric stereochemistry; moreover, thesugars can have (R) or (S) relative configurations, can exist as the (+)or (−) isomer, and can exist in the D or L configuration. The terms“anomer” and “anomeric” refer to the stereochemical configuration at theacetal, hemiacetal, or ketal carbon atom, as is well known in the art.

As used herein, “chitosan” refers to a polysaccharide polymer, eitherobtained from a natural source such as chitin, or syntheticallyprepared. Chemically, chitosan is predominantly a polymer ofβ-1,4-linked 2-amino-2-deoxyglucose monomers. When prepared from anatural source, the usual natural source is chitin, a major constituentof the shells of crabs, shrimp and other arthropods. Chitin ischemically a polymer comprising β-1,4-linked 2-acetamino-2-deoxyglucosemonomers. After isolation of chitin from its natural source, it istreated in a manner as to cause hydrolysis of the acetamido groupwithout cleavage of the sugar-sugar bonds, typically through alkalinehydrolysis. Chitosan is not a single molecular entity, but comprisespolymeric chains of various lengths.

As used herein, an “alkylated chitosan” is a material formed of chitosanmolecules to which carbon-containing molecules have been bonded. Theterm “alkylated chitosan” thus comprises a large number of possiblechemical structures, but they all share the unifying feature thatchemical bonds have been formed between the components of the chitosanmolecules and at least one carbon atom in each of the molecules that arebonded to the chitosan. For example, methylation of chitosan, in whichbonds are formed between methyl radicals or groups and atoms within thechitosan molecule, such as nitrogen, oxygen or carbon atoms, provides analkylated chitosan within the definition used herein. Othercarbon-containing groups may likewise be chemically bonded to chitosanmolecules to produce an alkylated chitosan. Specific examples includepoly(oxyalkylene)chitosan, wherein poly(oxyethylene), orpolyethyleneglycol, chains are covalently bonded to the chitosanbackbone, as well as acrylated chitosans, formed by alkylation ofchitosan with acrylates.

When referring to the “molecular weight” of a polymeric species such asan alkylated chitosan, a weight-average molecular weight is beingreferred to herein, as is well known in the art.

A “degree of substitution” of a polymeric species refers to the ratio ofthe average number of substituent groups, for example an alkylsubstituent, per monomeric unit of the polymer as defined.

A “degree of polymerization” of a polymeric species refers to the numberof monomeric units in a given polymer molecule, or the average of suchnumbers for a set of polymer molecules.

A “poly(oxyalkylene)chitosan” is a variety of alkylated chitosan asdefined herein. A “poly(oxyalkylene)” group is a polymeric chain ofatoms wherein two carbon atoms, an ethylene group, are bonded at eitherend to oxygen atoms. The carbon atoms of the ethylene group maythemselves bear additional radicals. For example, if each ethylene groupbears a single methyl group, the resulting poly(oxyalkylene) group is apoly(oxypropylene) group. If the ethylene groups are unsubstituted, thepoly(oxyalkylene) group is a poly(oxyethylene) group. Apoly(oxyethylene) group may be of a wide range of lengths, or degrees ofpolymerization, but is of the general molecular formula of the structure[—CH₂—CH₂—O—CH₂—CH₂—O—]_(n), where n may range from about 3 upwards to10,000 or more. Commonly referred to as “polyethyleneglycol” or “PEG”derivatives, these polymeric chains are of a hydrophilic, orwater-soluble, nature. Thus, a poly(oxyalkylene)chitosan is a chitosanderivative to which poly(oxyalkylene) groups are covalently attached. Aterminal carbon atom of the poly(oxyalkylene) group forms a covalentbond with an atom of the chitosan chain, likely a nitrogen atom,although bonds to oxygen or even carbon atoms of the chitosan chain mayexist. Poly(oxyethylene)chitosan is often referred to as“polyethyleneglycol-grafted chitosan” or “PEG-chitosan” or“PEG-g-chitosan” or “PEG-grafted-chitosan.”

The end of the poly(oxyethylene) chain that is not bonded to thechitosan backbone may be a free hydroxyl group, or may comprise acapping group such as methyl. Thus, “polyethylene glycol” or“poly(oxyethylene)” or “poly(oxyalkylene)” as used herein includespolymers of this class wherein one, but not both, of the terminalhydroxyl groups is capped, such as with a methyl group. In a specificmethod of preparation of the poly(oxyethylene)chitosan, use of apolyethyleneglycol capped at one end, such as MPEG (methylpolyethyleneglycol) may be advantageous in that if the PEG is firstoxidized to provide a terminal aldehyde group, which is then used toalkylate the chitosan via a reductive amination method, blocking of oneend of the PEG assures that no difunctional PEG that may crosslink twoindependent chitosan chains is present in the alkylation reaction. It ispreferred to avoid crosslinking in preparation of thepoly(oxyethylene)chitosans of the present invention.

FIG. 1 provides an example of the chemical structure of a segment of apoly(oxyethylene)chitosan polymer.

An alkylated chitosan is also a chitosan to which othercarbon-containing molecules are linked. An “acrylated chitosan” as theterm is used herein is an alkylated chitosan wherein acrylates have beenallowed to react with, and form chemical bonds to, the chitosanmolecule. An acrylate is a molecule containing an α,β-unsaturatedcarbonyl group; thus, acrylic acid is prop-2-enoic acid. An acrylatedchitosan is a chitosan wherein a reaction with acrylates has takenplace. The acrylate may bond to the chitosan through a Michael additionof the chitosan nitrogen atoms with the acrylate. FIG. 4 provides anexample of the chemical structure of a segment of an acrylated chitosanpolymer.

As used herein, a “polybasic carboxylic acid” means a carboxylic acidwith more than one ionizable carboxylate residue per molecule. Thecarboxylic acid may be in an ionized or salt form within the meaning ofthe term herein. A dibasic carboxylic acid is a polybasic carboxylicacid within the meaning herein. Thus, adipic acid is a polybasiccarboxylic acid, having two ionizable carboxylate residues per molecule.Disodium adipate is a polybasic carboxylic acid within the meaning ofthe term herein. Alternatively, the polybasic carboxylic acid may havehundreds or thousands of ionizable carboxylate groups per molecule; forexample, hyaluronan, also known as hyaluronic acid, which is an acidicpolysaccharide, is a polybasic carboxylic acid within the meaningassigned herein. The hyaluronan or hyaluronic acid may be in an ionizedor salt form within the meaning used herein. Thus sodium hyaluronate isa polybasic carboxylic acid within the meaning of the term as usedherein. An example of the chemical structure of a segment of ahyaluronan polymer is shown in FIG. 2.

As used herein, the term “acidic polysaccharide” refers to polymericcarbohydrates comprising carboxylic acid groups. The polymericcarbohydrate can be naturally occurring, or can be synthetic orsemi-synthetic. Examples of acidic polysaccharides are hyaluronan andcarboxymethyl cellulose. Carboxymethylcellulose, as is well-known in theart, is prepared by reaction of cellulose with sodium chloroacetate, andthe product is believed to contain acidic carboxymethyl groupscovalently linked to the primary hydroxyl groups of the anhydroglucosemonomeric units that make up the cellulose molecule. An oxidizedhyaluronan, that is, hyaluronan that has been treated with an oxidizingagent, such as sodium periodate, that cleaves vicinal diol moieties andprovides aldehyde groups, is an acidic polysaccharide within the meaningherein, and is also an oxidized polysaccharide within the meaningherein.

The term “oxidized polysaccharide” refers to a polymeric carbohydrate,acidic or non-acidic, that has undergone treatment with an oxidizingreagent, such as sodium periodate, that cleaves vicinal diol moieties ofthe carbohydrate to yield aldehyde groups. An oxidized hyaluronan, thatis, hyaluronan that has been treated with an oxidizing agent, such assodium periodate, that cleaves vicinal diol moieties and providesaldehyde groups, is an example of an acidic polysaccharide within themeaning herein. An oxidized dextran, that is, dextran that has beentreated with an oxidizing agent, such as sodium periodate, that cleavesvicinal diol moieties and provides aldehyde groups, is another exampleof an oxidized polysaccharide within the meaning herein. Another exampleof an oxidized polysaccharide is an oxidized starch, that is, a starchthat has been treated with an oxidizing agent, such as sodium periodate,that provides aldehyde groups. It is believed that the aldehyde groupsof oxidized polysaccharides interact with the amino groups of analkylated chitosan in such a way as to markedly increase the viscosityof the mixture and cause gelation. While not wishing to be bound bytheory, it is believed that this intermolecular interaction takes placethrough the formation of imines, or Schiff bases, between the aminogroups and the aldehyde groups.

A “dehydrating reagent” as used herein refers to a molecular speciesthat takes up the elements of water from a reaction, serving to drive acoupling reaction by thermodynamic factors. A dehydrating reagent is ancompound that undergoes reaction of covalent bonds upon taking up theelements of water, as opposed to merely absorbing water into physicalparticles or the like. Preferably a dehydrating reagent is an organiccompound. A specific example of a dehydrating reagent is a carbodiimide,that takes up the elements of water and undergoes changes in covalentbonds to ultimately yield a urea derivative.

As used herein, a “carbodiimide” is a class of organic substancescomprising a R—N═C═N—R′ moiety. The R and R′ groups may be any organicradicals. For example, when R and R′ are cyclohexyl groups, thecarbodiimide is 1,3-dicyclohexylcarbodiimide, a dehydrating reagent wellknown in the art. A water-soluble carbodiimide is a carbodiimide thathas sufficient solubility in water to form a homogeneous solution atconcentrations suitable to carry out the gelation reaction as describedherein. Typically, a water-soluble carbodiimide contains an ionic group,such as an ammonium salt, to confer water-solubility upon the molecule.The water-soluble diimide EDCI is1-ethyl-3-N,N-dimethylaminopropylcarbodiimide.

A “carboxyl activating reagent” as used herein refers to a molecularspecies that interacts with a carboxyl group in such a way as to renderthe carbonyl of the carboxyl group more susceptible to nucleophilicattack, as by an amine to yield an amide. This activation may take placeby formation of a complex or by formation of a covalent intermediate. Aspecific example of a carboxyl activating reagent is an N-hydroxycompound that can form an N-hydroxy ester of the carboxylic acid group,increasing the reactivity of the carbonyl moiety to nucleophilicaddition of a molecular species such as an amine.

The term “N-hydroxy compound” refers to an organic compound comprising achemical bond between a hydroxyl group and a nitrogen atom. SpecificN-hydroxy compounds such as N-hydroxysuccinimide andN-hydroxybenztriazole (1-hydroxy benzotriazole) are well known in theart as reagents that form esters with carboxylic acid groups and serveto activate the carboxylic acid group in reactions with nucleophiles.

“Gelatin,” as the term is used herein, is a collagen-derived materialthat is about 98-99% protein by dry weight. The approximate amino acidcomposition of gelatin is: glycine 21%, praline 12%, hydroxyproline 12%,glutamate 10%, alanine 9%, arginine 8%, aspartate 6%, lysine 4%, serine4%, leucine 3%, valine 2%, phenylalanine 2%, threonine 2%, isoleucine1%, hydroxylysine 1%, methionine and histidine<1% and tyrosine<0.5%.

An “aqueous medium,” as the term is used herein, refers to a liquidmedium composed largely, but not necessarily exclusively, of water.Other components may also be present, such as salts, co-solvents,buffers, stabilizers, dispersants, colorants and the like.

As used herein, the act of “mixing between mutually coupled syringes”refers to a procedure wherein one syringe is partially filled with oneingredient, a second syringe is partially filled with a secondingredient, and the two syringes are coupled together as with a luerconnector such that the contents of the syringes are mixed by drawingthe contents of one syringe through the connector into the secondsyringe, then reciprocally expelling the contents of the second syringeback into the first syringe. This process may be repeated until adequatemixing is achieved.

A “suture” or the act of “suturing” refers to the physical joining oftwo separate masses of tissue with thread or fiber, or alternativelywith solid materials such as fabrics or plastic films on which anadhesive is disposed, whereby the physical joining serves to hold theseparate tissue masses in close physical proximity at least temporarily,such as for the period of time required for biological healing to occur.A “suture line” is a line of, for example, stitches of thread as is usedto close an incision at the end of a surgical procedure.

A “therapeutic agent” is any agent which serves to repair damage to aliving organism to heal the organism, to cure a malcondition, to combatan infection by a microorganism or a virus, to assist the body of theliving mammal to return to a healthy state. A “protective agent” is anyagent which serves to prevent the occurrence of damage to an organism,such as by preventing the establishment of an infection by amicroorganism, to prevent the establishment of a malcondition, topreserve an otherwise healthy body in the state of health. Therapeuticand protective agents comprise pharmaceuticals, radiopharmaceuticals,hormones or their analogs, enzymes, materials for genetic therapy suchas antisense nucleotides or their analogs, macroscopic ingredients suchas bone powder as is used to induce bone growth, growth factors as maybe used to stimulate tissue growth such as by angiogenesis, or any othersuch agents as are medically advantageous for use to treat apathological condition. As used herein, “treating” or “treat” includes(i) preventing a pathologic condition from occurring (e.g. prophylaxis);(ii) inhibiting the pathologic condition or arresting its development;(iii) relieving the pathologic condition; and/or (iv) diminishingsymptoms associated with the pathologic condition.

A therapeutic agent or a protective agent may comprise a “drug.” As usedherein, a “drug” refers to a therapeutic agent or a diagnostic agent andincludes any substance, other than food, used in the prevention,diagnosis, alleviation, treatment, or cure of a disease. Stedman'sMedical Dictionary, 25^(th) Edition (1990). The drug can include anysubstance disclosed in at least one of: The Merck Index, 12^(th) Edition(1996); Pei-Show Juo, Concise Dictionary of Biomedicine and MolecularBiology, (1996); U.S. Pharmacopeia Dictionary, 2000 Edition; andPhysician's Desk Reference, 2001 Edition.

Specifically, the drug can include, but is not limited to, one or morepolynucleotides, polypeptides, oligonucleotides, gene therapy agents,nucleotide analogs, nucleoside analogs, polynucleic acid decoys,therapeutic antibodies, anti-inflammatory agents, blood modifiers,anti-platelet agents, anti-coagulation agents, immune suppressiveagents, anti-neoplastic agents, anti-cancer agents, anti-cellproliferation agents, and nitric oxide releasing agents.

The polynucleotide can include deoxyribonucleic acid (DNA), ribonucleicacid (RNA), double stranded DNA, double stranded RNA, duplex DNA/RNA,antisense polynucleotides, functional RNA or a combination thereof. Inone embodiment, the polynucleotide can be RNA. In another embodiment,the polynucleotide can be DNA. In another embodiment, the polynucleotidecan be an antisense polynucleotide.

The polynucleotide can be a single-stranded polynucleotide or adouble-stranded polynucleotide. The polynucleotide can have any suitablelength. Specifically, the polynucleotide can be about 2 to about 5,000nucleotides in length, inclusive; about 2 to about 1000 nucleotides inlength, inclusive; about 2 to about 100 nucleotides in length,inclusive; or about 2 to about 10 nucleotides in length, inclusive.

An antisense polynucleotide is typically a polynucleotide that iscomplimentary to an mRNA, which encodes a target protein. For example,the mRNA can encode a cancer promoting protein i.e., the product of anoncogene. The antisense polynucleotide is complimentary to the singlestranded mRNA and will form a duplex and thereby inhibit expression ofthe target gene, i.e., will inhibit expression of the oncogene. Theantisense polynucleotides of the invention can form a duplex with themRNA encoding a target protein and will disallow expression of thetarget protein.

A “gene therapy agent” refers to an agent that causes expression of agene product in a target cell through introduction of a gene into thetarget cell followed by expression. An example of such a gene therapyagent would be a genetic construct that causes expression of a protein,such as insulin, when introduced into a cell. Alternatively, a genetherapy agent can decrease expression of a gene in a target cell. Anexample of such a gene therapy agent would be the introduction of apolynucleic acid segment into a cell that would integrate into a targetgene and disrupt expression of the gene. Examples of such agents includeviruses and polynucleotides that are able to disrupt a gene throughhomologous recombination. Methods of introducing and disrupting geneswithin cells are well known to those of skill in the art.

Nucleotide and nucleoside analogues are well known on the art. Examplesof such nucleoside analogs include, but are not limited to, Cytovene®(Roche Laboratories), Epivir® (Glaxo Wellcome), Gemzar® (Lilly), Hivid®(Roche Laboratories), Rebetron® (Schering), Videx® (Bristol-MyersSquibb), Zerit® (Bristol-Myers Squibb), and Zovirax® (Glaxo Wellcome).See, Physician's Desk Reference, 2001 Edition.

As used herein, a “peptide” and a “protein” refer to polypeptides,linear polymers of amino acids, the difference between the terms“peptide” and “protein” largely being in the length of the polymer. Inone embodiment, the polypeptide can be an antibody. Examples of suchantibodies include single-chain antibodies, chimeric antibodies,monoclonal antibodies, polyclonal antibodies, antibody fragments, Fabfragments, IgA, IgG, IgM, IgD, IgE and humanized antibodies. In oneembodiment, the antibody can bind to a cell adhesion molecule, such as acadherin, integrin or selectin. In another embodiment, the antibody canbind to an extracellular matrix molecule, such as collagen, elastin,fibronectin or laminin. In still another embodiment, the antibody canbind to a receptor, such as an adrenergic receptor, B-cell receptor,complement receptor, cholinergic receptor, estrogen receptor, insulinreceptor, low-density lipoprotein receptor, growth factor receptor orT-cell receptor. Antibodies of the invention can also bind to plateletaggregation factors (e.g., fibrinogen), cell proliferation factors(e.g., growth factors and cytokines), and blood clotting factors (e.g.,fibrinogen). In another embodiment, an antibody can be conjugated to anactive agent, such as a toxin or a radionuclide.

An “anti-cancer agent” means an agent that either inhibits the growth ofcancerous cells, or causes the death of cancerous cells. Anti-canceragents include, e.g., nucleotide and nucleoside analogs, such as2-chloro-deoxyadenosine, adjunct antineoplastic agents, alkylatingagents, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites,hormonal agonists/antagonists, androgens, antiandrogens, antiestrogens,estrogen & nitrogen mustard combinations, gonadotropin releasing hotmone(GNRH) analogues, progestrins, immunomodulators, miscellaneousantineoplastics, photosensitizing agents, and skin & mucous membraneagents. See, Physician's Desk Reference, 2001 Edition.

An “antimicrobial,” as used herein, refers to a molecular entity that iseffective as a therapeutic agent or as a protective agent against aninfection by a microorganism, which could be a bacterium, a protozoan, afungus, a virus, or another pathogenic living organism. An antimicrobialmay be an antibiotic, effective against bacteria, includingaminoglycoside antibiotics such as gentamicin or streptomycin, acephalosporin such as cephalexin or cephtriaxone, a carbacephem such asloracarbef, a glycopeptide such as vancomycin, a macrolide such aserythromycin, a penicillin such as amoxicillin or ampicillin, apolypeptide such as bacitracin or polymyxin B, a quinolone such asciprofloxacin, a tetracycline such as oxytetracycline, a sulfonamide, orany other medically approved agent for treatment of bacterialinfections. Alternatively the antimicrobial may be an antifungal agentsuch as ketoconazole, miconazole or amphotericin B, or an antiviralagent such as acyclovir or AZT.

A “radioactive material” as used herein refers to any naturallyoccurring or manmade substance that emits ionizing radiation such asgamma rays, beta particles, Auger electrons, X-rays, or alpha particles.A radioactive material may be used for diagnostic purposes, such as forimaging as in positron emission tomography (PET). A radionuclidecommonly used for imaging diagnostics is fluorine-18. Alternatively aradioactive material may be used for therapeutic purposes, as intreating tumors. Radionuclides used therapeutically includetechnetium-99m, iodine-123 and -131, and gallium-67, among others.

In the claims provided herein, the steps specified to be taken in aclaimed method or process may be carried out in any order withoutdeparting from the principles of the invention, except when a temporalor operational sequence is explicitly defined by claim language.Recitation in a claim to the effect that first a step is performed thenseveral other steps are performed shall be taken to mean that the firststep is performed before any of the other steps, but the other steps maybe performed in any sequence unless a sequence is further specifiedwithin the other steps. For example, claim elements that recite “firstA, then B, C, and D, and lastly E” shall be construed to mean step Amust be first, step E must be last, but steps B, C, and D may be carriedout in any sequence between steps A and E and the process of thatsequence will still fall within the four corners of the claim.

Furthermore, in the claims provided herein, specified steps may becarried out concurrently unless explicit claim language requires thatthey be carried out separately or as parts of different processingoperations. For example, a claimed step of doing X and a claimed step ofdoing Y may be conducted simultaneously within a single operation, andthe resulting process will be covered by the claim. Thus, a step ofdoing X, a step of doing Y, and a step of doing Z may be conductedsimultaneously within a single process step, or in two separate processsteps, or in three separate process steps, and that process will stillfall within the four corners of a claim that recites those three steps.

Similarly, except as explicitly required by claim language, a singlesubstance or component may meet more than a single functionalrequirement, provided that the single substance fulfills the more thanone functional requirement as specified by claim language.

DETAILED DESCRIPTION

A hydrogel for use as a tissue sealant according to the presentinvention is a hydrogel that achieves a gelled state after formationfrom a premix composed of more than a single component. The hydrogel,which may be used to seal the tissues of a living mammal such as a humanpatient, is formed upon gelation of the premix, which is in the physicalform of a sol. Mixing of the components that make up the premix providesa liquid or semi-liquid sol that may be pumped or transferred by anytechnique suitable for handling somewhat viscous liquid materials, suchas syringes, pipettes, tubing and the like. Upon standing, the premixsol after a period of time sets up into the hydrogel of the presentinvention.

The premix sol and the resulting hydrogel that forms from the sol aresuitable for contact with living biological tissue, being biocompatibleand preferably biodegradable. Thus, the hydrogel can remain in contactwith living biological tissue within a human patient for an extendedperiod of time without damaging the tissue on which it is disposed. Inone specific embodiment, the hydrogel has adhesive properties towardsliving tissues on which it is disposed. In another specific embodiment,the hydrogel contains therapeutic or protective agents that are releasedinto the surrounding tissues on which the hydrogel is disposed. Inanother specific embodiment, the hydrogel has both adhesive propertiestowards the tissue on which it is disposed and also contains therapeuticor protective agents that are released into the surrounding tissues onwhich the hydrogel is disposed. In another specific embodiment thehydrogel contains microspheres or nanospheres containing therapeuticagents or protective agents that further control the release of theagents from the hydrogel.

A specific embodiment of a premix that forms a hydrogel according to thepresent invention comprises an alkylated chitosan. Referring to FIG. 1,in a specific embodiment an alkylated chitosan comprises apoly(oxyethylene)chitosan. The poly(oxyethylene)chitosan is a polymerformed of 2-amino-2-deoxyglucose monomeric units. Each monomeric unitcomprises a single free amino group and two free hydroxyl groups. InFIG. 1, one amino group is alkylated on the nitrogen atom with apoly(oxyethylene) chain, also known as a polyethyleneglycol chain. Inthe example provided in FIG. 1, the chitosan has a degree ofsubstitution of 0.5, because two of the four amino groups in thetetrameric unit shown bears the substituent. However, apoly(oxyethylene)chitosan according to the present invention may have adegree of amino group substitution ranging down to about 0.1 (whereinonly one in about every ten monomeric units is alkylated). Furthermore,a poly(oxyethylene)chitosan according to the present invention may alsobear the poly(oxyethylene) derivative on one of the two free hydroxylgroups in a given monomeric unit, or may comprises a mixture of N- andO-alkylated chitosan monomeric units, or be di-alkylated ortri-alkylated on a single monomer unit. Thus, a fully alkylated chitosanmonomeric unit has a degree of substitution of 3.0, and apoly(oxyethylene)chitosan according to the present invention may have adegree of substitution ranging up to 3.0 without departing from theprinciples of the invention.

A preferred degree of substitution for a poly(oxyethylene)chitosan isabout 0.35 to about 0.95. A particularly preferred degree ofsubstitution is about 0.5.

It should be understood that other poly(oxyalkylene) groups may besubstituted for the poly(oxyethylene) group shown in FIG. 1. Forexample, a poly(oxypropylene)chitosan may be used in place of, or inaddition to, the poly(oxyethylene)chitosan. A poly(oxypropylene) groupis the structure that would be obtained if the poly(oxyethylene) groupas shown in FIG. 1 bore a methyl group on every ethylene unit(—O—CH₂CH(CH₃)—O), or alternatively, every ethylene unit shown in FIG. 1were a 3-carbon linear propylene group (—O—CH₂CH₂CH₂—O—).

The number of monomeric units that make up a chitosan according to thepresent invention may vary widely without departing from the principlesof the invention. Any sample that contains more than a single moleculeof a chitosan derivative will almost inevitably contain a distributionof molecules of different molecular weights. A preferredpoly(oxyethylene)chitosan according to the present invention has amolecular weight of about 200 kD to about 600 kD.

In a specific embodiment, a premix for a hydrogel contains a polybasiccarboxylic acid comprising a hyaluronan. A member of the class of acidicpolysaccharides, a hyaluronan bears an ionizable carboxylic acid groupon every other monosaccharide residue. The hyaluronan can be in the formof a hyaluronate, that is, with at least most of the carboxylic acidgroups being in the ionized or salt form. Sodium hyaluronate is aspecific example. Referring to FIG. 2, a hyaluronan or a hyaluronic acidis a polybasic carboxylic acid, and the number of ionizable carboxylategroups per hyaluronan molecule is dependent on the degree ofpolymerization of the hyaluronan. The degree of substitution ofcarboxylic acid groups on the polymer backbone, assuming a monomericunit comprising the disaccharide formed of one glucuronic acidmonosaccharide and one 2-acetamido-2-deoxyglucose monosaccharide, is1.0. Every monomeric unit (disaccharide unit) bears a single ionizablecarboxylic acid group. A hyaluronan may be of any of a wide range ofdegrees of polymerization (molecular weights), but a preferredhyaluronan has a molecular weight of about 2,000 kD to about 3,000 kD.An example of a premix comprising a hyaluronan further comprises apoly(oxyethylene)chitosan, synonymously a PEG-chitosan.

Another specific embodiment of a premix that forms a hydrogel accordingto the present invention comprises an acrylated chitosan. Referring toFIG. 4, in a specific embodiment an alkylated chitosan comprises aacrylated chitosan wherein at least some of the free amino groups of the2-amino-2-deoxyglycose monosaccharide monomeric units are substitutedwith acrylate groups. It is believed that acrylate groups are bonded tofree amino groups of the chitosan via a Michael type conjugate additionwherein the nucleophilic amino group forms a bond to the β-carbon of theα,β-unsaturated acrylate, but the acrylate may be bonded to the chitosanin a different manner without departing from the principles of theinvention. Furthermore, as is illustrated in FIG. 4, acrylates maythemselves oligomerize after initial alkylation of the chitosanbackbone. The three-carbon carboxylic acid substituent on the leftillustrates the alkylation of chitosan with a single molecule ofacrylate, whereas the six-carbon dicarboxylic acid substituent on theright illustrates the product resulting from addition of a secondacrylate molecule to the first acrylate molecule, either prior to orsubsequent to addition of the first acrylate molecule to the chitosanbackbone.

A preferred degree of substitution of the chitosan backbone withacrylate groups according to the present invention is about 0.25 toabout 0.45. The number of monomeric units that make up a acrylatedchitosan according to the present invention may vary widely withoutdeparting from the principles of the invention. Any sample that containsmore than a single molecule of a chitosan derivative will almostinevitably contain a distribution of molecules of different molecularweights. A preferred acrylated chitosan has a molecular weight of about200 kD to about 600 kD.

A premix that includes an acrylated chitosan can also include apolybasic carboxylic acid comprising a dicarboxylic acid. A preferreddicarboxylic acid is a dicarboxylic acid wherein the two carboxylategroups are bonded to a moiety of about one to about twelve carbon atoms,which may comprise chains, aliphatic or aromatic rings, or heteroatomssuch as nitrogen, oxygen or sulfur. Referring to FIG. 5, a particularlypreferred dicarboxylic acid is a linear alkyl α,ω-dicarboxylic acid,which can crosslink acrylated chitosan polymer chains through theintermolecular formation of amide bonds between the chitosan aminogroups and the carboxylic acid groups of the dicarboxylic acid. Specificexamples of linear alkyl α,ω-dicarboxylic acids are malonic, succinic,glutaric, adipic, pimelic, suberic, azaleic, and sebacic acid. Aparticularly preferred example is adipic acid. A specific example of apremix of the invention comprises acrylated chitosan, disodium adipate,a dehydrating reagent and a carboxyl activating reagent.

In another specific embodiment, a premix that includes an alkylatedchitosan also includes a polybasic carboxylic acid comprising acarboxymethylcellulose. A carboxymethylcellulose is a derivative ofcellulose (a β-1,4 linked polymer of glucose) wherein hydroxyl groupsare substituted with carboxymethyl (—CH₂CO₂H) moieties. It is understoodthat the term carboxymethylcellulose comprises salts ofcarboxymethylcellulose, such as the sodium salt. A specific example of apremix comprises acrylated chitosan, carboxymethylcellulose sodium salt,a dehydrating reagent and a carboxyl activating reagent.Carboxymethylcellulose, as is well-known in the art, may have varyingdegrees of substitution, a “degree of substitution” referring to thenumber of derivatizing groups, herein carboxymethyl, per each monomerunit on the average. A particularly preferred carboxymethylcelluloseaccording to the present invention has a degree of substitution of about0.7 and a molecular weight of about 80 kD.

A premix according to the present invention comprises an aqueous medium.An aqueous medium necessarily includes water, and may include othercomponents including salts, buffers, co-solvents, additionalcross-linking reagents, emulsifiers, dispersants, electrolytes, or thelike.

A premix according to the present invention can comprise a dehydratingreagent. A preferred dehydrating reagent is a dehydrating reagent thatis sufficiently stable when dissolved or dispersed in an aqueous mediumto assist in driving the formation of the amide bonds before it ishydrolyzed by the water in the aqueous medium. A particularly preferredtype of dehydrating reagent is a carbodiimide, which is transformed to aurea compound through incorporation of the elements of water. An exampleof a water-soluble carbodiimide, is1-ethyl-3-(N,N-dimethylpropyl)carbodiimide (EDCI), which is preferred asit is soluble in the aqueous medium and thus does not require aco-solvent or dispersant to distribute it homogeneously throughout thepremix. Other water-soluble carbodiimides are also preferred dehydratingreagents.

A premix according to the present invention can comprise a carboxylactivating reagent. A preferred carboxyl activating reagent is a reagentthat serves to activate a carboxyl group towards formation of a newbond, such as an amide or ester bond with an amine or a hydroxyl-bearingcompound respectively. A specific embodiment of a carboxyl activatingreagent reacts with the carboxyl group to form a new compound as anintermediate, which then further reacts with another substance such asan amine to form an amide, or a hydroxyl-bearing compound to form anester. A preferred carboxyl activating reagent is an N-hydroxy compound.An N-hydroxy compound reacts with a carboxyl group to form an N-hydroxyester of the carboxylic acid, which may subsequently react with, forexample, an amino group to form an amide. An example of an N-hydroxycompound is N-hydroxysuccinimide. Another example of an N-hydroxycompound is N(1)-hydroxybenzotriazole.

Another preferred carboxyl activating reagent is a carbodiimide. Acarbodiimide reacts with a carboxyl group to form an O-acylisourea,which may subsequently react with, for example, an amine to form anamide, releasing the carbodiimide transformed through covalent additionof the elements of water to a urea compound. A preferred carbodiimide isa water-soluble carbodiimide, for example EDCI.

In a specific embodiment of the present invention, a carbodiimide mayserve both as a dehydrating reagent and as a carboxyl activatingreagent. Thus, a premix comprising an alkylated chitosan, a polybasiccarboxylic acid, and a carbodiimide is a specific embodiment accordingto the present invention. Another specific embodiment is a premixcomprising an alkylated chitosan, a polybasic carboxylic acid, acarbodiimide, and another molecular species wherein that species is acarboxyl activating reagent. Another specific embodiment is a premixcomprising an alkylated chitosan, a polybasic carboxylic acid, acarbodiimide, and another molecular species wherein that species is adehydrating reagent.

In an embodiment of a composition of the invention, an alkylatedchitosan forms a hydrogel after mixing with an oxidized polysaccharidein an aqueous medium. The initial sol formed after mixing undergoesgelation over a period of time, typically a few minutes, to provide ahydrogel of the invention. A specific example of an oxidizedpolysaccharide is oxidized dextran. Additional examples are oxidizedstarch and oxidized hyaluronan. A specific composition of the inventioncomprises acrylated chitosan and oxidized dextran, which readily forms ahydrogel without addition of a dehydrating reagent or of a carboxylactivating reagent.

Upon standing at room temperature or at the temperature of the humanbody, a premix sol of the invention, after a period of time that istypically in the order of a few minutes, for example about 0.5 minutesto about 20 minutes at about 37° C., or about 1 minute to about 10minutes at about 37° C., undergoes gelation to form a hydrogel of theinvention. A premix sol and a resulting hydrogel that forms from the solare suitable for contact with living biological tissue, beingbiocompatible and preferably biodegradable. Thus, the hydrogel canremain in contact with living biological tissue within a human patientfor an extended period of time without damaging the tissue on which itis disposed.

In an embodiment of the invention, the hydrogel contains therapeutic orprotective bioactive agents that are released into the surroundingtissues. Bioactive agents can be combined with premix solutions. bysimply blending commercially available solutions of polypeptides orother agents with the aqueous solutions, with gentle mixing. Cells canlikewise be blended with the composition, preferably immediately priorto emplacement to enhance survival of living cells.

A radiopaque material that is optionally incorporated in the compositionmay be fine particles of a selected radiopaque metal, such as gold,platinum, tantalum, barium, or the like. Alternatively, a radiopaqueagent can be an iodinated organic compound. A specific example isiohexol.

A variety of types of a bioactive agent can be incorporated into thecomposition of the invention. The agent can be a molecular entity, suchas a regenerative agent such as one or more human growth modulatingfactors such as interleukins, transformation growth factor-b, fibroblastgrowth factor (FGF) or vascular endothelial growth factor (VEGF), a genetherapy agent, a cogener of platelet derived growth factor, a monoclonalantibody directed against growth factors; a drug, or a cell regenerationfactor. A bioactive agent may also be a cellular entity such as aplurality of drug-producing cells or of regenerative cells such as stemcells.

In another embodiment of the invention, the hydrogel containsmicrospheres or nanospheres containing therapeutic agents or protectivebioactive agents, the microspheres or nanospheres further controllingthe release of the bioactive agents from the hydrogel into thesurrounding tissues. A “microsphere” or a “nanosphere” as used herein isa particulate body of dimensions of the order of microns (micrometers)or nanometers respectively, wherein the particulate body may be hollowor solid. Microspheres and nanospheres may be formed of organic orinorganic materials. For example, a nanosphere may comprise abuckminsterfullerene (buckyball), which is organic. Alternatively ananosphere may comprise microporous glass, which is inorganic. It isunderstood that the terms encompass solid lipid nanoparticles, whereinthe nanosphere particles are formed from a solid lipid. Preferably themicrosphere or the nanosphere contains a drug or other substance, thetiming of the release of which it is advantageous to control.

Due to the abundance of cationic amino groups in the chitosan structure,it is known that drugs with carboxyl groups can been conjugated theretoand sustained release can be achieved through the hydrolysis of theamide or ester bonds linking drugs to the chitosan molecule. Y. D.Sanzgiri, et al., Pharm. Res., 1, 418 (1990). As a polyelectrolyte,chitosan can also electrostatically conjugate sensitive bioactive agentswhile preserving their bioactivities and enhancing their stabilities.Such derivatives may be formed with the acrylated chitosan of thepresent invention, and will likewise serve to provide for sustainedrelease and to preserve the bioactivity and to enhance the stability ofthe conjugated agent(s).

The abundance of positive charges on the alkylated chitosan enables theelectrostatic binding of biologically active proteins such as rhVEGF.This is the most gentle mode of conjugating proteins and thus protectingand preserving the bioactivity of sensitive proteins like rhVEGF. Theconjugation of proteins like rhVEGF to the alkylated chitosan alsoserves as a mechanism for modulating the biological activity of thegrowth factor, thereby limiting the potential for induction ofuncontrolled tissue development.

The types of cells that may be incorporated into the composition includeprogenitor cells of the same type as those from the tissue site, andprogenitor cells that are histologically different from those of thetissue site such as embryogenic or adult stem cells, that can act toaccelerate the healing process. The therapeutic composition comprisingcells can be administered in the form of a solution or a suspension ofthe cells mixed with the polymer solution, such that the cells aresubstantially immobilized within the application site upon gelation ofthe premix. This serves to concentrate the effect of the therapeuticagent or the cells at the site of the tissue damage and to provide forrelease of the agent or of the cells or of cellular products over acourse of time.

A hydrogel of the invention adheres to the living tissues on which it isdisposed such that it may be used as a tissue sealant. A tissue sealantof the invention has at least sufficient adhesivity such that aftergelation, the hydrogel resists detachment from the tissue when subjectedto a force at least as strong as may be applied when a patient moves, orby the weight of an organ acted on by gravity, or by involuntary motionsof surrounding tissues (heartbeat, peristalsis, etc.). For applicationswhere the disrupting strain is likely to be more severe, for example ina limb where voluntary muscles exert a greater force, the tissue sealantpreferably adhesively seals the tissue such that it can resist arelatively large disruptive force. The hydrogel is of sufficientstrength and elasticity such that the physical integrity of the mass ofsealant as it is disposed on the tissue is maintained while the hydrogeladheres to the tissue. The inventive tissue sealant thus is suitable forat least temporarily holding the tissue surrounding a tear, hole,perforation, incision or any separation of tissue in physical proximity.For example, the inventive tissue sealant is adapted to seal anylaceration, cut, abrasion, or disruption of dermal tissue, i.e., skin.

A specific tissue sealant of the invention also preferably seals thetear, hole, perforation or incision in order to prevent leakage of anyvital bodily fluids that are normally retained by the tissue in itsundamaged state. For example, the outer membrane surrounding the brainand the spinal cord, the dura mater, serves to contain the cerebrospinalfluid in which the nervous system tissue is normally immersed. Aspecific use for the tissue sealant according to the present inventionis the repair or sealing of the dura, such as after brain surgery. Tosurgically reach the brain in order to carry out any of the operativeprocedures that may be applied within the brain to treat or cure amalcondition, an incision must be made in the dura, and when thesurgical procedures within the brain are complete, it is desirable toclose the dura as tightly as possible to avoid leakage of cerebrospinalfluid to areas external to the dura as well as to allow healing of thetissue. A specific tissue sealant of the present invention is wellsuited to provide this closure and sealing, either as a reinforcement ofa suture line or without additional closure techniques being used.

In this specific use of a tissue sealant of the invention, the premix isprepared and is applied to the incision in the sol form. For aid to thesurgeon in visualizing the application of the tissue sealant, thesealant may comprise a dye or a fluorescent material to better enablethe surgeon to see and thus control the distribution of the sealant onthe tissue being repaired. The sealant may also comprise a radio-opaqueagent to aid in visualization of the disposition of the sealantpost-operatively.

A specific technique for preparing the premix is with the use of twosyringes coupled with a Luer coupling fitting. The fitting optionallycontains a three-way T-valve. One syringe is partially filled with asolution comprising an alkylated chitosan dissolved in an aqueousmedium. A second syringe is partially filled with a solution comprisinga polybasic carboxylic acid dissolved in an aqueous medium. Optionally,either solution may contain additional components such as a co-solvent,dispersant, emulsifier or other additive to assist in dissolving ordispersing the polymeric component. Optionally, either syringe mayfurther contain a dehydrating reagent, a carboxyl activating reagent orboth.

Formation of the premix takes place by reciprocally exchanging thecontents of the two syringes through the coupling fitting, firstdepressing the plunger of one syringe to expel the contents into thesecond syringe, then depressing the plunger of the second syringe toexpel the mixed contents back into the first syringe. This procedure ispreferably repeated until substantial homogeneity is achieved.Optionally a dehydrating reagent, a carboxyl activating reagent, orboth, may be added by replacing the empty syringe with a third syringecharged with a solution of the reagent(s) and repeating the reciprocalexchange. Then, the charged syringe containing the homogeneous premix isattached to a suitable application tip, which is used to transfer thepremix such that it is disposed on the tissue to be sealed, for examplean incision in the dura mater.

In a similar manner the premix may be applied to other tissue types inneed of sealing, such as dermal tissue, musculature, and so forth. Forexample, the premix of a tissue sealant may be applied to seal theannulus of a ruptured inter-vertebral disc as part of a surgicalprocedure to repair a ruptured disc.

A premix may be mixed up in any suitable container, taken up in asyringe or pipette, and transferred to the tissue to be sealed, or itmay be poured onto the tissue in a controlled manner. Preferably,complete application of the premix to the tissue takes place beforesignificant gelling occurs, although the premix may undergo a certainamount of thickening prior to application without departing from theprinciples of the invention.

The premix preferably gels within a relatively short time frame, but notinstantly, to enable the premix to be applied to the tissue to be sealedbefore substantial gelling takes place. Nevertheless, the premixpreferably forms the hydrogel within the timeframe of minutes, such thatsurgical procedures are not unduly delayed or interrupted by periods oftime wherein the gelling is taking place. Preferably gelling takes placewithin about 1 minute to about 12 minutes at the body temperature of thepatient, around 37° C. It is understood that even after the premix gelsto a sufficient degree that the hydrogel ceases to be flowable,additional gelling or hardening may take place. It is preferred thatgelation has occurred to a sufficient degree within the 1 to 12 minutetime window to permit surgical procedures to continue. Thus, it ispreferred that both gelling and adhesion of the hydrogel be sufficientlyachieved in this time window that the tissue and its surroundings may beat least gently manipulated by the end of the time window withoutcausing flow of ungelled premix or detachment of adhered hydrogel fromthe tissue on which the hydrogel is disposed.

The tissue sealant of the present invention preferably is dimensionallystable after gelling in the presence of aqueous media, such as in aliving human body. The tissue sealant does not absorb water or swellgreatly in the presence of water. This is particularly preferable whenthe tissue sealant is disposed in proximity to nerves, where swellingand the resulting pressure applied to the nerves is particularlydisadvantageous. The dimensional stability of a tissue sealantcomprising a hydrogel is an outstanding feature according to the presentinvention. This dimensional stability enables a tissue sealant of thepresent invention to be particularly well-suited for use in proximity toneural tissue, for example in sealing of dura mater in brainneurosurgery. In brain neurosurgery, avoidance of post-surgical swellingis especially critical, thus the dimensional stability of a tissuesealant of the present invention is highly advantageous in such use.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the claims. Other aspects,advantages, and modifications are within the scope of the claims andwill doubtless be apparent to persons of ordinary skill in the art.

EXAMPLES Example 1 Preparation of Acrylated Chitosan

5.52 ml of acrylic acid was dissolved in 150 ml of double distilledwater and 3 g of chitosan (Kraeber® 9012-76-4, molecular weight 200-600kD) was added to it. The mixture was heated to 50 C and vigorouslystirred for 3 days. After removal of insoluble fragments bycentrifugation, the product was collected and its pH was adjusted to 11by adding NaOH solution. The mixture was dialyzed extensively to removeimpurities.

Example 2 Preparation of PEG-Chitosan

Monomethyl-PEG-aldehyde was prepared by the oxidation of Monomethyl-PEG(MPEG) with DMSO/acetic anhydride: 10 g of the dried MPEG was dissolvedin anhydrous DMSO (30 ml) and chloroform (2 ml). Acetic anhydride (5 ml)was introduced into the solution and the mixture is stirred for 9 h atroom temperature. The product was precipitated in 500 ml ethyl ether andfiltered. Then the product was dissolved in chloroform andre-precipitated in ethyl ether twice and dried.

Chitosan (0.5 g, 3 mmol as monosaccharide residue containing 2.5 mmolamino groups, Kraeber 9012-76-4, molecular weight 200-600 kD) wasdissolved in 2% aqueous acetic acid solution (20 ml) and methanol (10ml). A 15 ml sample of MPEG-aldehyde (8 g, DC: 0.40) in aqueous solutionwas added into the chitosan solution and stirred for 1 h at roomtemperature. Then the pH of chitosan/MPEG-monoaldehyde solution wasadjusted to 6.0-6.5 with aqueous 1 M NaOH solution and stirred for 2 hat room temperature. NaCNBH₃ (0.476 g, 7.6 mmol) in 7 ml water was addedto the reaction mixture dropwise and the solution was stirred for 18 hat room temperature. The mixture was dialyzed with dialysis membrane(COMW 6000-8000) against aqueous 0.5 M NaOH solution and wateralternately. When the pH of outer solution reached 7.5, the innersolution was centrifuged at 5,000 rpm for 20 min. The precipitate wasremoved. The supernatant was freeze-dried and washed with 100 ml acetoneto get rid of unreacted MPEG. After vacuum drying, the final product(white powder) was obtained as water soluble or organic solvent solublePEG-g-Chitosan. The yield of water soluble derivatives was around 90%based on the weight of starting chitosan and PEG-aldehyde.

Example 3 Preparation of a PEG-Chitosan and Hyaluronan Hydrogel

Hyaluronan (sodium hyaluronate, Kraeber 9067-32-7) was dissolved inwater as a 0.5% solution by weight. PEG-chitosan, prepared as describedin Example 2, was dissolved in water as a 5% solution by weight. Asample of each solution (0.5 mL of each) was mixed, then a solution ofEDCI (20 μL of a solution in water at 350 mg/mL) was added and thesolution was thoroughly mixed. Immediately a solution ofN-hydroxysuccinimide (20 μL of a solution in water at 125 mg/mL) wasadded and thoroughly mixed in to form a premix. The premix gelled into ahydrogel in about 7 minutes at ambient temperature (22° C.). At 37° C.gelation occurred in about 2 minutes.

Example 4 Preparation of a Acrylated Chitosan and Adipic Acid Hydrogel

A sample of acrylated chitosan prepared as described in Example 1 wasdissolved in water at a concentration of 2% by weight. A sample of thissolution (0.5 mL) was mixed with a solution of adipic acid in water (40μL of a 20 mg/mL solution), then a solution of EDCI (20 μL of a 350mg/mL solution) and the solution thoroughly mixed. Then, a solution ofN-hydroxysuccinimide in water (20 μL of a 125 mg/mL solution) was mixedin. The premix gelled in about 9 minutes at ambient temperature (22°C.). At 37° C. gelation occurred in about 3 minutes.

Example 5 Preparation of an Acrylated Chitosan andCarboxymethylcellulose Hydrogel

A sample of acrylated chitosan prepared as described in Example 1 wasdissolved in water at a concentration of 2% by weight. A sample ofcarboxymethylcellulose sodium salt (Polysciences no. 06140, MW 80 kD,degree of substitution 0.7) was dissolved in water at a concentration of5% by weight. These two solutions (0.25 mL each) were mixed with asolution of EDCI (20 μL of a 6.5% solution) and the solution thoroughlymixed. Then, a solution of N-hydroxysuccinimide in water (20 μL of a 35%solution) was mixed in. The solution gelled in about 10 minutes atambient temperature (22° C.).

Example 6 Preparation of Oxidized Dextran

Dextran (5 g) was dissolved in 400 mL of distilled H₂O, then 3.28 g ofNaIO₄ dissolved in 100 mL ddH₂O was added. The mixture was stirred at25° C. for 24 hrs. 10 ml of ethylene glycol was added to neutralize theunreacted periodate following by stirring at room temperature for anadditional hour. The final product was dialyzed exhaustively for 3 daysagainst doubly distilled H₂O, then lyophilized to obtain a sample ofpure oxidized dextran.

Example 7 Analyses of Oxidized Dextran

The degree of oxidation of the oxidized dextran was determined byquantifying the aldehyde groups formed using t-butyl carbazate titrationvia carbazone formation. A solution of oxidized dextran (10 mg/ml in pH5.2 acetate buffer) was prepared; and a 5-fold excess tert-butylcarbazate in the same buffer was added and allowed to react for 24 hrsat ambient temperature, then a 5-fold excess of NaBH₃CN was added. After12 hrs, the reaction product was precipitated three times with acetoneand the final precipitate was dialyzed thoroughly against water,followed by lyophilization. The degree of oxidation (i.e., abundance ofaldehyde groups) was assessed using ¹H NMR by integrating the peaks: 7.9ppm (proton attached to tert-butyl) and 4.9 ppm (anomeric proton ofdextran).

Example 8 Preparation of an Oxidized Dextran/Acrylated Chitosan Gel

A 1 mL sample of a 1-3% aqueous oxidized dextran in water solution wasmixed with 1 mL of a 1-3% aqueous acrylated chitosan solution. Themixture was gently stirred for 10 seconds. Gelation occurred within 30seconds to 10 minutes at temperatures ranging from 5° C. to 37° C.

Example 9 Preparation of Oxidized Hyaluronan

Sodium hyaluronate (1.0 gram) was dissolved in 80 ml of water in a flaskshaded by aluminum foil, and sodium periodate (various amounts)dissolved in 20 ml water was added dropwise to obtain oxidizedhyaluronan (oHA) with different oxidation degrees. The reaction mixturewas incubated at ambient temperature and 10 ml of ethylene glycol wasadded to neutralize the unreacted periodate following by stirring atroom temperature for an additional hour. The solution containing theoxidized hyaluronan was dialyzed exhaustively for 3 days against water,then lyophilized to obtain pure product (yield: 50-67%).

Example 10 Analyses of Oxidized Hyaluronan

The degree of oxidation of oxidized hyaluronan was determined byquantifying aldehyde groups formed with t-butyl carbazate titration viacarbazone formation. A solution of the oxidized hyaluronan (10 mg/ml inpH 5.2 acetate buffer) and a 5-fold excess tertbutyl carbazate in thesame buffer were allowed to react for 24 hrs at ambient temperature,followed by the addition of a 5-fold excess of NaBH₃CN. After 12 hrs,the reaction product was precipitated three times with acetone and thefinal precipitate was dialyzed thoroughly against water, followed bylyophilization. The degree of oxidation (i.e., abundance of aldehydegroups) was assessed using ¹H NMR by integrating the peaks: 1.32 ppm(tert-butyl) and 1.9 ppm (CH₃ of hyaluronic acid).

Example 11 Preparation of a PEG-Chitosan/Hyaluronan Hydrogels

A solution of PEG-chitosan (2.5 wt %) and hyaluronan (0.5%) in water atpH in the range of about 3.5 to about 5.5 was made up by first makingsolutions of the two polymers independently at the stated pH, thenmixing the solutions. The mixture rapidly formed a viscous solution.

A solution of PEG-chitosan (1.3% w/v, 25 g), hyaluronan (2% w/v, 2.3 g),and 0.1 M HCl (pH 1.45, 0.45 g) was made up. The viscosity increasedmarkedly within about 30 seconds. The pH of the resulting mixture waswithin the 3.5 to 5.5 range.

Example 12 Preparation of a Gelatin/Oxidized Hyaluronan Hydrogel

A 20% w/v solution of gelatin in water (1 ml) was mixed with a 20%solution of partially oxidized Hyaluronan (1 ml) (20.3% oxidized). Thesolution was warmed to about 40-45° C., above the melting point of thegelatin, and was mixed. At about 37° C. gelation occurs in about 15minutes. Optionally, a buffer system of 0.1 M borax at pH 9.4 may beused.

Example 13 Application of a Premix to Canine Pericardium In Vitro

A premix comprising an alkylated chitosan, a polybasic carboxylic acid,a dehydrating reagent and a carboxyl activating reagent in water wasmade up using two syringes joined by a Luer connector. The somewhatviscous but flowable composition was then applied to a square (about 5×5cm) of damp canine pericardium in which an incision had previously beenmade. The clear viscous liquid was allowed to stand in place for severalminutes. A hydrogel formed and adhered to the tissue such that thepericardium square could be picked up, manipulated and stretched,showing that a clear, elastic hydrogel had sealed the incision. Thehydrogel was probed with blunt forceps tips but did not rupture ordetach from the surrounding tissue.

Example 14 PEG-Chitosan/Hyaluronan Hydrogel Adhesion to Murine Muscle

A premix formed as in Example 13 was disposed on the muscle tissue of adead mouse. A hydrogel was completely formed after about 10 minutes. Amoderate amount of force applied with a pair of forceps did not eitherdetach the hydrogel from the muscle, or rupture the hydrogel. A highdegree of hydrogel elasticity was observed.

Example 15 PEG-Chitosan/Hyaluronan Hydrogel Sealing of Full-ThicknessDermal Wounds in Mouse In Vivo

A wound completely penetrating the thickness of the dermal layer of aliving mouse was provided. A premix prepared as in Example 13 wasdisposed in the wound. After one week, the hydrogel was observed to bein place and detachment had not occurred. Evidence of healing along theoriginal wound bed was observed.

Example 16 Evaluation of PEG-Chitosan/Hyaluronan Hydrogel Adhesion andSealing in Canine Dura Mater In Vitro

A premix formed as in Example 13 was applied to a sample of canine duramater bathed in saline at 37° C. The sealant formed a hard and durablesubstance within about 90 to 120 seconds of injection. The sample ofdura mater was picked up with forceps and gentle pressure was applied tothe edges. There was no tearing of the tissue seam.

1. A biocompatible, biodegradable composition for use in sealing abiological tissue of a living mammal, the composition comprising ahydrogel, the hydrogel being produced by mixing a first solutioncomprising an alkylated chitosan with a second solution comprisingoxidized dextran, wherein the mixture, when applied to a biologicaltissue in need of sealing, forms a hydrogel within a minute after mixingthereby sealing the biological tissue.
 2. (canceled)
 3. The compositionof claim 1 wherein the hydrogel is adapted to adhere to biologicaltissue of a living mammal, to adhesively seal the tissue.
 4. (canceled)5. The composition of claim 1 wherein the alkylated chitosan comprisesacrylated chitosan. 6-11. (canceled)
 12. The composition of claim 1wherein the living mammal is a living human being. 13-14. (canceled) 15.The composition of claim 1 further comprising a therapeutic orprotective agent. 16-25. (canceled)
 26. A method of preparing acomposition of claim 1 for use in sealing a biological tissue of aliving mammal, the method comprising: combining with mixing a firstsolution comprising an alkylated chitosan and a second solutioncomprising oxidized dextran to form the mixture; then, applying themixture to biological tissue; and then, allowing the mixture to form thehydrogel by gelation such that the hydrogel formed by gelation isdisposed on the biological tissue.
 27. (canceled)
 28. The method ofclaim 26 wherein the hydrogel formed by gelation from the mixtureadheres to the biological tissue on which it is disposed, to adhesivelyseal the tissue.
 29. (canceled)
 30. The method of claim 26 wherein thealkylated chitosan comprises an acrylated chitosan. 31-34. (canceled)35. The method of claim 26 further comprising dissolving the alkylatedchitosan in the aqueous medium at a concentration of about 1% to about10% by weight.
 36. The method of claim 35 wherein the alkylated chitosanconcentration is about 3% to about 7% by weight.
 37. The method of claim35 wherein the alkylated chitosan concentration is about 5% by weight.38-46. (canceled)
 47. A method of sealing a disrupted biological tissuein need thereof in a living mammal using the biocompatible,biodegradable composition of claim 1, comprising applying the mixture tothe disrupted biological tissue.
 48. The method of claim 47 wherein thebiological tissue comprises dura mater.
 49. The method of claim 47wherein the biological tissue comprises brain or spinal cord. 50-53.(canceled)
 54. The method of claim 47 wherein the biological tissuecomprises neural tissue or dermal tissue.
 55. (canceled)
 56. The methodof claim 47 comprising adhesively sealing the disrupted biologicaltissue against leakage of a biological fluid. 57-60. (canceled)